During the current funding period of this grant we have established that Interleukin 1 (IL-1) and Tumor necrosis factor (TNF), two potent stimulators of bone resorption released in greater amounts from bone marrow monocytes after ovariectomy (ovx), play a critical role in inducing bone loss and stimulating bone resorption in ovx rats and mice. This was accomplished by demonstrating that treatment with the IL-1 inhibitor, IL-1 receptor antagonist (IL-1ra) and the TNF inhibitor, TNF binding protein (TNFbp) decrease bone loss in ovx animals and do so by inhibiting osteoclastogenesis. We now plan to investigate the mechanism by which IL-1 and TNF stimulate osteoclastogenesis in conditions of estrogen deficiency. Since we have also found that stromal cells from ovx mice constitutively produce higher amounts of M-CSF, a factor critical for the proliferation and differentiation of the hemopoietic osteoclast precursor, in Specific Aim # 1 we will test the hypothesis that in ovx mice the increased production of IL-1 and TNF is responsible for the observed selection of a high M-CSF-producing stromal cell phenotype. This will be accomplished by determining whether IL-1ra and TNFbp prevent the effects of ovx on the constitutive production of M-CSF from long term stromal cell cultures purified from the bone marrow of ovx mice treated in vivo with IL-1ra and TNFbp. In Specific Aim # 2 we will investigate the mechanism by which stromal cells from ovx mice constitutively produce more M-CSF than stromal cells from sham operated mice. This will be accomplished by determining if ovx increases the fraction of bone marrow stromal cell which produce M- CSF, by investigating the mechanism by which the M-CSF gene is constitutively activated in stromal cells from ovx mice and by determining if ovx increases the expression of M-CSF receptor in stromal cells. Since we have found that estrogen not only indirectly condition the secretory characteristic of the stromal cell, but also directly regulate their M-CSF mRNA steady state expression, in Specific Aim # 3 we will determine the mechanism by which estrogen regulates the expression of the M-CSF gene in stromal cells. The potential significance of this project is high as it may 1) clarify the mechanism by which the increased production of IL-1 and TNF induced by ovx stimulates osteoclastogenesis and 2) elucidate the mechanism of the direct inhibitory effects of estrogen on the M-CSF gene, an additional important mechanism by which the hormone regulates osteoclastogenesis and bone resorption.